Method for transforming plant, the resultant plant and gene thereof

ABSTRACT

A method for achieving the sufficient expression of a gene in a useful higher plant which has been transformed by transferring the above gene encoding a protein having a function carried by another organism so as to impart the function to the plant. Namely, a method for transforming a useful plant by transferring a gene of another species into the plant characterized in that the region of a factor relating to the poly(A) addition of the mRNA of the useful plant to be transformed contained in the base sequence of the gene of the other species is denatured into another base sequence not relating to the poly(A) addition of the mRNA without substantially altering the function of the protein encoded by the gene to be transferred; and a gene usable in the gene transfer.

TECHNICAL FIELD

[0001] This invention relates to a method for transforming a usefulplant by introducing a gene of another species into the useful plant.More particularly, the present invention pertains the method fortransforming the useful plant characterized in that the region of afactor relating to the poly (A) addition of the mRNA of the useful plantto be transformed contained in the base sequence of the gene of theother species is modified into another base sequence not relating to thepoly (A) addition of the mRNA without substantially altering thefunction of the protein encoded by the gene to be introduced, the usefulplant produced by it, a nucleic acid in which base sequence used theretois modified, and a method for producing the said nucleic acid.

BACKGROUND ARTS

[0002] Growth of plants needs great numbers of nutrients. The plantsabsorb most of these nutrients necessary for growth from roots. Theplants, which can not absorb nutrients in soil due to having hereditarylow enzyme activities required for absorption of nutrients, are known.

[0003] For example, iron is an essential element for almost organisms,and is essentially required for large numbers of enzymes involved infunctioning cells such as photosynthesis and respiration. Ironsolubilized in soil exists mainly in the form of Fe(III) chelate [insome case, Fe(II) chelate]. In general, Fe(II) is prevalently absorbedas compared with Fe(III) by plants, but the absorption depends on plantspecies.

[0004] Plants have two types of mechanisms of iron uptake, i.e.absorption mechanism (I) (refer to FIG. 1) and absorption mechanism (II)(refer to FIG. 2) (Mori, 1994).

[0005] The absorption mechanism (I) shown in FIG. 1 consists of: (1)release of proton into the rhizosphere (Olsen and Brown, 1980), (2)increased reducing activity of Fe(III) in cell membrane of roots (Brownet al., 1961 and Chaney et al., 1972), and (3) excretion of reduced andchelating substances from roots (Hether et al., 1981). Namely, Fe(III)is chelated by the released chelating substance, and Fe(III)-chelate inthe free space of roots is reduced to Fe(II) on the cell membrane byferric-chelate reductase and is absorbed through Fe(II) transporter. Itis also thought that the proton is released into the rhizosphere andactivity of reductase is increased by lowering pH in the free space.However, the problem is known that since reducing activity of Fe(III) isinhibited by higher pH, strong pH buffering action due to highconcentration of carbonate anion results to cause lime chlorosis(Marchner et al., 1986).

[0006] Absorption mechanism (II) shown in FIG. 2 is specific to grassand is consisting of: (1) synthesis of mugineic acids(phytosiderophore), (2) release of mugineic acids into the rhizosphere,(3) formation of soluble complex of iron and mugineic acids, and (4)absorption of mugineic acids-iron complex by plants body (Takagi, 1976and Takagi et al., 1984). The iron uptake mechanism by such theabsorption mechanism (II) observed in grass has advantage not to beinhibited by higher pH.

[0007] Yeast (Saccharomyces cerevisiae), a model organism of eukaryote,performs iron absorption similar to the above absorption mechanism (I).Since in studies on the gene level in the higher plants, Fe(II)transporter has cloned by complementation of iron absorption mutant ofyeast (Eida et al., 1996), no detailed mechanism of iron absorption hasbeen studied.

[0008] Contrary to that, the mechanism in yeast (Saccharomycescerevisiae) has been studied in detail. Absorption of iron in yeast isinitiated by a reduction of Fe(III) to Fe(II) by ferric-chelatereductase FRE1 and FRE2 (Dancis et al., 1990,1992, Georgatsou andAlexandraki, 1994). In the mechanism for uptake of reduced Fe(II) intocells, high affinity absorption mechanism and low affinity absorptionmechanism are known.

[0009] In the absorption of iron by the high affinity absorptionmechanism, after reoxidation of Fe(II) by multicopper oxidase FET3(Askwith et al., 1994), Fe(III) may be incorporated into cells by ferrictransporter FTR1 (Stearman et al., 1996). Copper is required in thereoxidation of divalent iron (Dancis et al., 1994, Klomp et al., 1997),and copper supplying pathway to FET3 has also studied (Yuan et al., 1995and Lin et al., 1997).

[0010] Absorption of iron by the low affinity absorption mechanism maybe performed by an action of Fe(II) transporter FET4 (Dix et al., 1994,1997).

[0011] Such the iron absorption mechanism in yeast may be applied toplants, and plants which can be grown in the iron deficient soil may becreated.

[0012] For that purpose, we have created transgenic tobacco, to whichFRE1 gene of yeast provided by Dr. Dancis (NIH) was transformed(Yamaguchi, 1995).

[0013] However, in the transgenic tobacco, to which FRE1 gene wastransformed, the reducing activity was not changed as compared with thatof wild type. As a result of Northern hybridization analysis, thetranscriptional product of yeast gene FRE1 in tobacco was so small as0.9 kb.

[0014] Example of such incomplete transcription, in which gene ofanother species is transformed into the higher plant, is gene group Cryencoding δ-endotoxin (insecticidal protein) of Bacillus thuringiensis.More than 42 Cry genes have been known and are classified into 4 classes(cryI-cryIV)(Whiteley and Schnepf, 1986). The gene encoding thisinsecticidal protein was introduced into the higher plant, but neitherexpression nor extremely low expression was found.

[0015] This may be caused by (1) difference in codon usage, (2) high ATcontent in Cry gene, (3) unstable in mRNA, and (4) a partial splicing ofCry gene as intron.

[0016] A preparation of the transgenic plant with high expression ofprotein has been reported. Namely, in order to express Cry gene groupefficiently in the higher plant, base sequence of Cry gene is modifiedto arrange with base sequence of the plant, and the primer issynthesized, then is completely synthesized by PCR (Perlak et al., 1991,Fujimoto et al., 1993, and Nayak et al., 1997).

[0017] Although transformation of the higher plant by introducing geneof the another organism species has known, the expression thereof wasnot sufficient. Various reasons have been provided as described in theabove.

[0018] We have made extensive studies on factors for achieving thesufficient expression of a gene in a higher plant which has beentransformed by introducing the above gene encoding a protein having afunction carried by another organism so as to impart the function in theuseful higher plant, and found that base sequence of the factor relatingto the poly(A) addition of the mRNA of the transformed plant is animportant part of the expression.

[0019] Consequently, the present invention provides a method forexpressing the introduced gene in the transgenic higher plant with highefficiency, the said transgenic higher plant, and a method for modifyinggene therefor.

DISCLOSURE OF THE INVENTION

[0020] The present invention relates to a method for transforming auseful plant by introducing another gene into the useful plantcharacterized in that the region of a factor relating to the poly(A)addition of the mRNA of the useful plant to be transformed contained inthe base sequence of the said another gene is modified into another basesequence not relating to the poly(A) addition of the mRNA withoutsubstantially altering the function of the protein encoded by the geneto be introduced. Example of the region of a factor relating to thepoly(A) addition of the mRNA is preferably AATAAA like base sequence,further the said region of a factor relating to the poly(A) addition ofthe mRNA is preferably the region existing downstream of the GT-richbase sequence. Further, modification of the base sequence of the saidregion is preferably carried out based on the codon usage of thetransformed useful plant.

[0021] In the method of the present invention, it is preferable thatbase G and T rich region in the gene to be introduced is small;difference in content of base G and C within whole region of the gene tobe introduced is small; the sequence has no ATTTA sequence; and/orupstream of the initiation codon of the gene to be transferred has Kozaksequence.

[0022] Further, the present invention relates to the transformed usefulplant, which can be produced by the method of the present invention. Thetransformed useful plant of the present invention can be the organismand the seed and has no limitation in the form.

[0023] Further, the present invention relates to a nucleic acid,especially DNA, having the modified base sequence, which can be used bythe above transforming method.

[0024] The base sequence of the nucleic acid of the present invention isa modified base sequence which can be expressed in the transformeduseful plant with high efficiency, and, for example, is a factorrelating to the poly(A) addition of mRNA of the said useful plant, andis characterized in that a part of factor relating to the said poly(A)addition is replaced by the other base sequence, further the said basesequence has small G- and T-rich region of the base in the gene to beintroduced, has small difference between G- and C-content of the basethroughout the gene to be introduced, has no ATTTA sequence and/orpreferably the upstream of the initiation codon of the gene to beintroduced has Kozak sequence.

[0025] Further, the present invention relates to a method for productionof the above nucleic acids characterized in that the above nucleic acidsare divided into several fragments and these fragments are ligated.

BRIEF DESCRIPTION OF DRAWINGS

[0026]FIG. 1: Absorption mechanism (I) of iron in plants.

[0027]FIG. 2: Absorption mechanism (II) of iron in plants.

[0028]FIG. 3: A position of poly(A) addition in higher plant.

[0029]FIG. 4: G- and T-rich sequence in yeast gene FRE1.

[0030]FIG. 5: Schematic illustration of refre1 synthesis.

[0031]FIG. 6: Sequence of 30 primers used in the synthesis of refre1.

[0032]FIG. 7: Relationship between refre1 sequence and primer.

[0033]FIG. 8: Preparative scheme of full length refre1.

[0034]FIG. 9: Total sequence of designed refre1.

[0035]FIG. 10: Graphical illustration of G- and T-contents in FRE1(upper level) and refre1 calculated by continued 8 base unit.

[0036]FIG. 11: Structure of binary vector pRF1.

[0037]FIG. 12: A photograph showing growth of transgenic plant of thepresent invention.

[0038]FIG. 13: A photograph showing anthesis of transgenic plant of thepresent invention.

[0039]FIG. 14: Result of Southern hybridization of the transformantusing refre1 as a probe.

[0040] Left: Digestion by EcoRI and HindIII

[0041] Right: Digestion by HindIII

[0042] No.1-No.2: Transformant

[0043] W.T: wild type

[0044]FIG. 15: Result of Northern hybridization of the transformantusing refre1 as a probe.

[0045] No.1-No.2: Transformant

[0046] W.T: wild type.

[0047]FIG. 16: A photograph showing activity of ferric-chelate reductasein roots indicating red coloring of BPDS-Fe(II) complex by Fe(II).

[0048] Left: Wild type showing no red coloring.

[0049] Right: Transformant showing red coloring in roots.

[0050]FIG. 17: Photograph showing replicate experiment of the same as inFIG. 16 using another transformant. Red coloring is observed in thetransformant (right).

[0051]FIG. 18: Photograph showing activity of ferric-chelate reductaseby red coloring of BPDS-Fe(II) complex in roots, using second generationof plant obtained from seeds of the transformant. Red coloring ofBPDS-Fe(II) complex is observed in the second generation of thetransformant deft).

BEST MODE FOR CARRYING OUT THE INVENTION

[0052] The useful plants transformed in the present invention are nolimitation, if these are industrially used plants such as foods andpharmaceuticals, and are preferably higher plants such as grains,vegetables, fruits and tobacco.

[0053] Another gene introduced in the present invention is not limited,if it is useful for plants and has no detrimental effects for plants andhuman. It may be directly useful gene for plants and gene providingresistance against chemicals such as herbicide, and is preferably enzymederived from organisms such as bacteria and yeast. For example,ferric-chelate reductase FRE1 of yeast involving absorption of iron ispreferable.

[0054] We have found that in a transformed plant, factors affectingexpression of introduced gene may be a base sequence which determinesaddition of poly(A) of mRNA. Further, we have found that in the upstreamof the base sequence, which defines addition of poly(A), GT-rich basesequence is necessary. Namely, in the presence of GT-rich base sequence,addition of poly(A) is determined in plants, subsequently mRNA is splitat the position after 10-30 bp from the poly(A) signal, for exampleAATAAA like base sequence, then poly(A) is added by an action of poly(A)polymerase. Accordingly, in case that the introduced gene has such thebase sequence, in the transgenic plants, full length mRNA can not beexpressed, and mRNA is split in the position after 10-30 bp from thepoly(A) signal having AATAAA like base sequence.

[0055] Consequently, the present invention is characterized in that thepoly(A) signal of plant in the introduced gene, for example AATAAA likebase sequence, preferably GT-rich base sequence, is modified to anotherbase sequence.

[0056] A method of design for modifying base sequence is, at first,codon is selected for not to change amino acid sequence encoded by geneto be introduced. Amino acid sequence can be changed, if the sequencehas not substantial effect for function for protein, preferably theamino acid sequence may not be changed.

[0057] In case that multiple numbers of codons, which encode an aminoacid, is known, the codon having high rate of usage in the plant ispreferably selected by considering the codon usage of the plant to betransformed.

[0058] Further, not only modification of base sequence of poly(A) signalbut also deletion of GT-rich base sequence is preferable. Especially, incase that GT-rich base sequence exists with high proportion, sincepossibility of splitting mRNA in the region of poly(A) signal like basesequence appearing in the downstream of the GT-rich base sequence ishigh, a modification for reducing amount of GT content in such theregion is important.

[0059] Further, in the present invention, in addition to the abovemodification, it is preferable to modify in order to make smallerdifference between G- and C-content of bases throughout the full regionof gene to be introduced. More preferably, the sequence should notcontain ATTTA sequence, which is known as unstable sequence of mRNA,and/or the sequence has Kozak sequence, which is known as a sequence foreffective translation of mRNA in the eukaryote, in the upstream of theinitiation codon of gene to be introduced.

[0060] The method of the present invention includes a modification ofbase sequence combined further with usual method of modification to theabove modification of base sequence.

[0061] The method for modification of base sequence can be made withoutlimitation by known various methods. For example, any conventionalmethod of modification by point mutation and splitting with restrictionenzyme can be applied.

[0062] Further, in case that large numbers of base have to be modifiedor gene itself to be introduced has short length, it can be prepared bysynthesis. As explained later concretely, even if length of gene islong, the gene is divided into several fragments, and each fragment,which is amplified by PCR, is ligated using restriction enzymes, thengene having modified base sequence can be prepared.

[0063] The method of the present invention is further explained moreconcretely, but the method of the present invention can not be limitedwithin the scope of the following explanation, and the broad applicationthereof based on the said explanation can be performed by the personskilled in the art.

[0064] We have tried to study the reason why length of mRNA of yeastFRE1, which was introduced into tobacco, was short (0.9 kb). As for thereasons for incomplete length of transcriptional product of yeastferric-chelate reductase FRE1, which was introduced in tobacco, twopossibilities were considered, i.e.

[0065] (1) a part of mRNA was spliced as intron, and

[0066] (2) a transcription was terminated within coding region.

[0067] As a result of further analysis by RT-PCR, it was found thatpoly(A) addition occurred within coding region in the transgenictobacco, to which FRE1 gene was transformed.

[0068] Example of the confirmed expression of yeast gene introduced intothe higher plant is invertase (Hincha, 1996). In the present experiment,new knowledge, in which these is a case that full length mRNA can not besynthesized even in the same eukaryotic gene by introducing FRE1 gene,could be obtained.

[0069] Reason why full length mRNA could not be synthesized in the FRE1transformed transgenic tobacco was addition of poly(A) within the codingregion of FRE1.

[0070] A poly(A) site is not limited within one position, and in theupstream of each poly(A) site, AAUAAA like base sequence, putativepoly(A) signal region was observed. However, although several AAUAAAlike sequences were observed at 5′-site of FRE1, the poly(A) additionwas not observed in these positions.

[0071] It may be a GU-rich sequence located in the upstream of thepoly(A) signal to determine addition of poly(A) in plant. Namely, ifGU-rich sequence exists, addition of poly(A) may be occured in theplant, and in the position of “PyA”, which is located at the distance of10-30 bp from the subsequently appeared AAUAAA like sequence, mRNA issplitted, then the poly(A) may be added by an action of poly(A)polymerase.

[0072] In conclusion, the fact that GU-rich sequence, which has norelation to addition of poly(A) in yeast, determines addition of poly(A)in plant, is a cause for not forming full length mRNA in the transgenictobacco, to which FRE1 is transformed.

[0073] A sequence of ferric-chelate reductase FRE1 having GT-rich regionis shown in FIG. 4. In FIG. 4, the boxed sequences are thought to beGT-rich regions.

[0074] As a result, in order to express ferric-chelate reductase FRE1 intobacco, deletion of GT-rich sequence from FRE1 gene maybe effective.However, at present, as for the sequence, which determines addition ofpoly(A) in plant, there may be only known that a consensus sequence maybe GU-rich and the sequence is not completely determined. So long as theexact consensus sequence has not be known, there may be possibility notto be obtainable the full length mRNA by only changing the sequence. Wehave, therefore, tried to design base sequence corresponding to codonusage of plants to be transformed without changing amino acid sequenceof FRE1 in order to synthesize full length mRNA in plant.

[0075] In order to express yeast ferric-chelate reductase in tobacco, wehave redesigned base sequence corresponding well to the codon usage oftobacco without changing amino acid sequence of FRE1. In design of basesequence, the following points are considered.

[0076] (1) GT-rich region is eliminated;

[0077] (2) Base sequence AATAAA, which may be a poly(A) signal, and thesimilar base sequence are eliminated;

[0078] (3) In order to confirm easily the base sequence, restrictionsites are set at the position in about every 400 bp (417-436 bp), andthe sequence is divided in 5 segments;

[0079] (4) Base sequence, ATTTA sequence (Ohme-Takagi, 1993), which iscalled as unstable sequence of mRNA, is eliminated;

[0080] (5) In order not to make difference between base content G and Cin whole region, position of codons are replaced; and

[0081] (6) Kozak sequence, which is a sequence for effectivelytranslating mRNA in eukaryote (Kozak, 1989) is attached prior to theinitiation codon.

[0082] The thus designed modified base sequence of yeast ferric-chelatereductase FRE1 is shown in Sequence listing, SEQ ID NO: 1. Amino acidsequence thereof is shown in SEQ ID NO: 2.

[0083] The designed gene is designated as reconstructed FRE1(hereinafter designates as “refre1”).

[0084] The refre1 of the present invention is synthesized by dividinginto 5 segments (A-E) as shown in FIG. 5.

[0085] A segment A consists of a sequence of 1-434 bp, in whichrestriction sites are designed as in base 1: EcoRI, base 7: XbaI andbase 429: BamHI.

[0086] A segment B consists of a sequence of 429-845 bp, in whichrestriction sites are designed as in base 429: BamHI and base 840: MroI.

[0087] A segment C consists of a segment of 840-1275 bp, in whichrestriction sites are designed as in base 840: MroI and base 1270: SaA.

[0088] A segment D consists of a segment of 1270-1696 bp, in whichrestriction sites are designed as in base 1270: SaA and base 1691: PstI.

[0089] A segment E consists of a segment of 1691-2092 bp, in whichrestriction sites are designed as in base 1691: PstI, base 2081: SacIand base 2087: HindIII.

[0090] Each segment A-E, each consisting of sequence having 417-436 bp,is synthesized using 6 primers having 77-83 mer, respectively. Thirtyprimers used, from A-1 to E-6, are shown in FIG. 6. These base sequenceare shown in sequence listings, SEQ ID NO: 5-SEQ ID NO: 34.

[0091] Among primers in the segments, −1, −2 and −3 are sense strands,and primers −4, −5 and −6 are anti strands. Primers are designed so asto have complementary base sequence consisting of 12 or 13 bp in the 3′end for primers −3 and −4, and overlapping sequence consisting of 12 or13 bp in 3′ end for primers −1 and −2, −2 and −3, −4 and −5, and −5 and−6. The primer −1 and −6 is designed to have restriction site at thebase 1 in 5′ end.

[0092] Relationship between these primers and the designed basesequences is shown in FIG. 7.

[0093] Respective segments A-E are prepared by PCR using primerssynthesized according to the above base sequence (refer to FIG. 5).

[0094] After the reaction mixture of third step PCR was electrophoresedwith 0.8% agarose gel, bands having expected length (417-436 bp) werecut and purified, then were cloned into plasmid pT7Blue (R) vector(supplied by Takara Corp.). The base sequences of the obtained cloneswere confirmed and the clones having exact base sequences were selectedby applying fluorescent DNA sequencer DSQ-1000L (made by ShimadzuCorp.).

[0095] Segments having exact sequences were obtained and full lengthrefre1 was prepared by applying restriction sites according to methodsshown in FIG. 8.

[0096] Direction of insertion in segments B and E is essentiallyrequired for preparation of full length. In other segments, the segmentscontaining exact base sequence were used without relation to directionof the insertion.

[0097] Total base sequence of the obtained refre1 is shown in FIG. 9.Specific features of sequence of refre1 are:

[0098] (1) 75.3% of homology to the original FRE1 (100% homology inamino acid sequence);

[0099] (2) To have no sequence consisting of only G or T which iscontinuously linked more than 8 bases;

[0100] (3) It does not contain not only a sequence AATAAA but alsosequences replaced by any one of bases in the above sequence;

[0101] (4) It does not contain a sequence ATTTA; and

[0102] (5) No difference is observed in GC content through the wholeregion of the sequence.

[0103]FIG. 10 shows decreased numbers of sequences consisting of serialG and T in refre1 as compared with the original FRE1. This illustratesGT content of serial 8 bases in the FRE1 and refre1 sequences. As shownin FIG. 10, uniformity of GT content in refre1 is demonstrated ascompared with the original FRE1.

[0104] The thus synthesized gene refre1 is introduced into tobacco(Nicotiana tabacum L. var. SRI). As a result of transformation, 68kanamycin resistant plants were reproduced. In order to confirmtransformation of the objective gene in the reproduced plant and itscopying number, genomic Southern hybridization was conducted. As aresult, one to several copied plants of the transformant, refre1 genewas confirmed.

[0105] A method from gene introduce into plant cells to reproduction ofplant can be performed by conventional method, for example, as describedin “Laboratory Manual on functional analysis of plant gene” (Maruzen)[ref. (4)].

[0106] Specifically, a fragment of restriction enzymes, XbaI and SacI,in refre1, which was cloned with pT7Blue(R) vector by the above method,was exchanged with ORF of β-glucronidase in the binary vector pBI121(TOYOBO Co. Ltd.) to prepare binary vector pRF1. The structure of thebinary vector pRF1 is shown in FIG. 11.

[0107] The thus obtained binary vector pRF1 was transformed into E.coli, and E. coli, which is bearing helper plasmid pRK2013, were shakecultured at 37° C. for overnight. On the other hand, Agrobacteriumtumefaciens C58 was shake cultured in LB liquid culture medium 1 mlcontaining proper antibiotic at 26° C. for 2 nights. Each 100 μl thereofwas mixed on LB plate without containing antibiotic, cultured at 26° C.for 2 nights, then surface of the plate was scraped by using platinumspatula, and cultured on the selection plate [LB plate containing 100μg/μl rifampicin (Rf) and 25 μg/μl kanamycin (Km)] at 26° C. for 2nights to form single colony.

[0108] The single colony was shake cultured in LB (Km and Rf) liquidmedium 4 ml at 26° C. for 2 nights. Plasmid was extracted, andrestriction enzyme treated cleavage pattern of the plasmid indicatedexistence of pRF1.

[0109] Plant to be transformed was prepared as follows.

[0110] Two or three young leaves, size about 8 cm, of wild type tobaccowere cut, and were sterilized in a petri dish, filled with sterilizedsolution (hypochlorous acid 10% and Tween 20, 0.1%), with stirring for15 minutes. After rinsing three times with sterilized water, the leaveswere cut off in 8 mm squares. To the leaves in a petri dish was addedthe cultured liquid 3 ml of the binary vector bearing Agrobacteriumtumefaciens C58, which was cultured 26° C. for 2 nights. Mter 1 minute,the liquid was rapidly removed by using Pasteur pipette, and residualliquid was removed off on the autoclaved filter paper.

[0111] Fragments of leaves were put on a culture medium, added withbenzyl adenine and naphthaleneacetic acid to the MS medium, and culturedunder light condition at 25° C. for 3 days. Thereafter, the fragments ofleaves were transferred to the medium added with CLAFORAN, and culturedfor 1 week further were transferred to the medium added with CLAFORANand kanamycin, then inoculated in every 2 weeks. When calli were inducedand shoots were formed, the shoots were cut off with scalpel and weretransferred to the MS medium added with kanamycin.

[0112] Shoots with roots were transplanted to the vermiculite and theplants were raised with supplying hyponex (Hyponex Japan Co. Ltd.) toobtain the transgenic plants.

[0113] Sixty-eight transgenic plants having kanamycin resistance couldbe obtained as a result of transformation. Example of photograph of thegrown plant is shown in FIG. 12 and the photograph of plant with floweris shown in FIG. 13.

[0114] Among them, 5 individual plants were treated with genomicSouthern hybridization. Result is shown in FIG. 14.

[0115] In the genomic Southern hybridization, extraction of genomic DNAfrom the transgenic tobacco was performed according to the descriptionin “Plant Cell Technology Series 2, Protocol for PCR Experiments ofPlants” (Shujun-Sha) [Ref. (2)]. The obtained genomic DNA was digestedby restriction enzymes EcoRI and HindIII and the hybridization wasperformed by using a probe, which was prepared with full length fragmentof refre1 as a template ([α-32P]-dATP was used).

[0116] In the genomic Southern hybridization shown in FIG. 14, amountsof DNA were arranged at the time of restriction enzyme treatment, butdeviation was observed due to treating with ethanol after restrictionenzyme treatment. Consequently, darkness of bands detected is not alwaysreflecting the copy numbers of the introduced gene.

[0117] In digestion with restriction enzymes, EcoRI and HindIII band,size 3.2 kb, which was expected in all individuals, was observed.However, in the individual No. 12, a band with slightly smaller than 3.2kb was detected. According to this result, 1 copy of refre1 in No. 1 andNo. 11, 3 or 4 copies in No. 2 and 4 copies in No. 9 were thought toexist.

[0118] In the digestion by HindIII on No. 12, band could not be detecteddue to loading failure on the gel.

[0119] As a result of the above genomic Southern hybridization analysis,the refre1 gene was found to be introduced into the selected fiveindividuals.

[0120] When the sequence is cleaved by restriction enzymes EcoRI andHindIII, a sequence from promoter to terminator is cleaved, and theintroduced refre1 gene is transcribed to mRNA under regulation ofCaMV35S promoter. In the EcoRI and HindIII digestion of No. 12, a reasonfor detecting a band slightly smaller than 3.2 kb might be due to thefact that one of the introduced construction was cleaved beforeintegration in the plant genom, and was inserted into the position closeto EcoRI or HindIII site in the plant genom.

[0121] Next, in the transformed tobacco No. 1 and No. 2, in which theintroduced refre1 gene was confirmed by the genomic Southernhybridization hereinbefore, formation of full length mRNA was confirmedby Northern analysis.

[0122] In the Northern analysis, a method of blotting was performed, forexample, according to the conventional method described in “Cloning andSequencing” (Noson-Bunka-Sha) [Ref. (1)], and the method inhybridization was performed according to the method as described inSouthern analysis hereinbefore.

[0123] A result of Northern hybridization is shown in FIG. 15. In FIG.15, no band is detected in the lane of wild type (W.T.). In lanes of No.1 and No. 2, major bands with a size of 2.5 kb are detected and severalbands smaller than that are detected.

[0124] In Northern analysis, formation of the full length mRNA as aresult of introducing refre1 of the present invention could beconfirmed. In the present analysis, fundamentally although total RNAshould be extracted from the root, in which mRNA is expected to beexpressed, in this experiment, if the root is cut, then the plant of thesubsequent generation can not be obtained, consequently the extractionhas to perform from leaves. Since CaMV35S as a promoter is used andrefre1 gene is expressed in the whole parts of plant, even in theanalysis performed by extraction of total RNA from leaves, it isconfirmed that the transcription can be performed in leaves and roots ofplants and site of addition of poly(A) is not changed.

[0125] As a result of Northern hybridization, since the transcriptionalproduct of 2.5 kb was confirmed in the tobacco, to which refre1 wasintroduced, poly(A) addition might occur by NOS terminator. Full lengthmRNA was also found in the tobacco, to which refre1 was introduced.

[0126] A band smaller than the length of 2.5 kb as seen in FIG. 15 isdetected at the position corresponding to rRNA as compared with that ofphotograph after electrophoresis. Although it was thought to be anonspecific absorption of probe in rRNA, since it was not hybridizedwith wild type RNA, it is surely hybridized with transcriptional productof refre1. There may be possibly produced the shorter mRNA than the fulllength mRNA in the refre1. However, since it is detected as the samelength with rRNA, it may be thought that, in the electrophoresis of RNA,mRNA of refre1 may be dragged by rRNA, which exists in large amount.This reason may be clarified by Northern hybridization with purifiedpoly(A)+RNA, however as obviously shown in FIG. 15, most of mRNA is fulllength mRNA and there is no reason to trace and clarify such the reason.

[0127] In order to perform Northern hybridization, RNA of thetransformant of No. 1, which was found as one copy, and that of thetransformants of No. 2, which were found as 3 or 4 copies, wereelectrophoresed, it was found that the bands of No. 2 were dark coloreddepending on copy numbers of refre1 gene.

[0128] Further, among the obtained transformed 68 plants of tobacco(selected by kanamycin), constant ferric-chelate reductase in root wasconfirmed in 6 plants.

[0129] For detection of reductase activity, a property of red colorformation of the complex of bathophenanthroline disulfonic acid (BPDS),which is a strong chelater for Fe(II), with Fe(II) was applied. Afterremoval of vermiculite from transformant and wild type tobacco, rootswere laid on the gel containing BPDS with shield light using aluminumfoil and stood at 27° C. for 24 hours. Reduction of Fe(III) wasconfirmed by coloring in the rhizosphere of the transformant.

[0130] Photographs confirming reductase activity are shown in FIG. 16and FIG. 17. In photographs of FIG. 16 and FIG. 17, red coloring isobserved in the transformant of the right photographs as shown withblack color.

[0131] As shown, ferric-chelate reductase was detected in all of 6plants (selected by kanamycin), which were used for confirmation offerric-chelate reductase activity in roots. In order to demonstratedifference between the transformed tobacco and wild type tobacco,reaction time of reductase was set for long time as 24 hours, but thedifference was observed at about 1 hour of the reaction time. In all of6 plants used in the transformation experiments, the leaves, which wereput on the gel for detecting activity, showed tendency of crinkle ascompared with the condition of wild type leaves. This may be due toinvolvement in the mechanism of the introduced refre1 gene expression inthe leaves. Though not so many times of activity tests were performedbecause of this phenomenon, it is clear that refre1 gene is expressed asa result of transcription and translation in the root under regulationof CaMV35S promoter.

[0132] As explained in the above, we have created novel tobacco whichcould express yeast ferric-chelate reductase FRE1 in the higher planttobacco.

[0133] Reasons for not obtaining full length transcription product ofdifferent organism gene may be due to two possibilities includingsplicing a part of mRNA as an intron, and adding poly(A) within thecoding region.

[0134] The present invention provides a method for designing basesequence for obtaining full length transcriptional product bytransferring gene of different species in the higher plant. In themethod of the present invention, in order to avoid addition of poly(A)in the coding region, it was found that it is necessary to design thesequence consisting of continued base sequence of 8 bases or morewithout containing sequence consisting of only G or T, and to design thesequence without containing not only a sequence of AATAAA but also asequence, in which any one of bases thereof is replaced by another base(i.e. NATAAA, ANTAAA, AANAAA, AATNAA, AATANA, or AATAAN).

[0135] It was also found that to design the sequence, in which G and Ccontents should be constantly distributed in the full region, isimportant.

[0136] Further, in the concrete explanation of the present inventionhereinbefore, since CaMV35S was used as a promoter, ferric-chelatereductase was expressed in the transformed tobacco of the whole plant.As shown, locally expressing gene can be expressed in the systemic plantas a result of combining with the promoter. On the contrary, theexpressing gene in the systemic plant can be expressed in the localregion by using combination with preferable promoter.

[0137] A mechanism of absorption by reduction of Fe(III) is specific toiron acquiring mechanism in the monocots and dicots except for grass,and also the grass may absorb iron by reducing Fe(III) to Fe(II) underthe condition of sufficient iron. As a result of ligating theferric-chelate reductase gene refre1 of the present invention with apromoter, which is specifically active in the root under iron deficientcondition, novel grass, in which iron absorption mechanism (I) andabsorption mechanism (II) under the condition of iron deficiency can befunctioned, may able to be created.

[0138] References in the present invention are listed hereinbelow.

[0139] (1) Cloning and Sequence (1989), Noson-Bunka-Sha

[0140] (2) Protocol of PCR Experiments for Plants (1995), Shujun-Sha

[0141] (3) Biological Experiments, Illustrated Fundamentals of GeneticAnalysis (1995), Shujun-Sha

[0142] (4) Labolatory Manual, Functional Analysis of Plant Genes (1992),Maruzen

[0143] (5) Askwith, C., et al., Cell 76: 403-410 (1994)

[0144] (6) Brown, J. C., et al., Soil Sci. 91: 127-132 (1961)

[0145] (7) Chaney, R. L., et al., Plant Physiol. 50: 208-213 (1972)

[0146] (8) Dancis, A., et al., Mol. Cell. Biol. 10: 2293-2301 (1990)

[0147] (9) Dancis, A., et al., Proc. Natl. Acad. Sci. USA 89: 3869-3873(1992)

[0148] (10) Dix, D. R., et al., J. Biol. Chem. 269: 26092-26099 (1994)

[0149] (11) Dix, D., et al., J. Biol. Chem. 272: 11770-11777

[0150] (12) Eide, D., et al., Proc. Natl. Acad. Sci. USA 93: 5624-5628(1996)

[0151] (13) Fujimoto, H., et al., Biol/Technology 11: 1151-1155 (1993)

[0152] (14) Gallie, D. R., et al., Plant Cell 9: 667-673 (1997)

[0153] (15) Georgatsou, E., et al., Mol. Cell. Biol. 14: 3065-3073

[0154] (16) Guo, Z., et al., Biochem. Sci. 21: 477-481 (1996)

[0155] (17) Hassett, R., et al., J. Biol. Chem. 270: 128-134 (1995)

[0156] (18) Hether, N. H., et al., J. Plant Nutr. 7: 667-676 (1984)

[0157] (19) Hincha, D. K., et al., J. Plant Physiol. 147: 604-610 (1996)

[0158] (20) Kozak, M., J. Cell Biol. 108: 229-241 (1989)

[0159] (21) Lin, S. J., et al., J. Biol. Chem. 272: 9215-9220 (1997)

[0160] (22) Marschner, H., et al., J. Plant Nutr. 9: 695-713 (1986)

[0161] (23) Mewes, H. W., et al., Nature 387: 7-8 (1997)

[0162] (24) Mori, S., (1994), Biochemistry of metal micronutrients inthe rizosphere. (Eds. Monthey, J. A., Crowley, D. E., Luster, D. G.,Lewis Publishers), pp225-249

[0163] (25) Naito, S., et al., Plant Mol. Biol. 11: 109-124 (1988)

[0164] (26) Nakanishi, H., et al., Plant Cell Physiol. 34: 401-410(1933)

[0165] (27) Nayak, P., et al., Proc. Natl. Acad. Sci. USA 94: 2111-2116(1997)

[0166] (28) Ohme-Takagi, M., et al., Proc. Natl. Acad. Sci. USA 90:11811-11815 (1993)

[0167] (29) Okumura, N., et al., J. Plant Nutr. 15: 2157-2172 (1992)

[0168] (30) Okumura, N., et al., Plant Mol. Biol. 25: 705-719 (1994)

[0169] (31) Olsen, R. A., et al., J. Plant Nutr. 2: 629-660 (1980)

[0170] (32) Perlak, F. J., et al., Proc. Natl. Acad. Sci. USA 88:3324-3328 (1991)

[0171] (33) Stearman, R., et al., Science 271: 1552-1557 (1996)

[0172] (34) Takagi, S., Soil Sci. Plant Nutr. 22: 423-433 (1976)

[0173] (35) Takagi, S., et al., J. Plant Nutr. 7: 469-477 (1984)

[0174] (36) Whiteley, H. R., et al., Annu. Rev. Microbiol. 40: 549-576(1986)

[0175] (37) Wu, L., et al., Plant J. 8: 323-329 (1995)

[0176] (38) Yuan, D. S., et al., Proc. Natl. Acad. Sci: USA 92:2632-2636 (1995)

EXAMPLES

[0177] The present invention will be explained in detail hereinbelow inexamples, but is not construed as limiting within these examples.

[0178] In the examples hereinbelow, fundamental gene manipulation isperformed according to the description of “Cloning and Sequencing”(Noson-Bunka-Sha) and analysis of base sequence of gene is performed byusing DNASIS (made by Hitachi Corp.).

Example 1 Extraction of Total RNA from FRE1 Introduced TransgenicTobacco

[0179] Extraction of total RNA from FRE1 introduced transgenic tobaccowas performed according to a method described in the reference (Naito etal., 1988).

[0180] Leaves 2 g of FRE1 introduced transgenic tobacco were put in themortar, and liquid nitrogen was added thereto, then leaves werecompletely mashed. Three fold amounts of buffer for extraction and equalamount of phenol/chloroform (1:1) were added to the debris andsuspended, then centrifuged at 8000 rpm for 15 minutes, and extractedwith chloroform once. Ethanol precipitation was conducted at −80° C. for30 minutes, and centrifuged at 4° C. for 30 minutes. Precipitate waswashed with 70% ethanol and dried in vacuum. The precipitate wasdissolved in DEPC treated water 1 ml, centrifuged at 13500 rpm for 3minutes, and the supernatant was transferred to a new tube, further 10 MLiCl, ¼ volume, was added and allowed to stand on ice for 2 hours. Themixture was centrifuged at 12000 rpm at 4° C. for 10 minutes, then theprecipitate was washed with 70% ethanol and dried in vacuo. The driedproduct was dissolved in DEPC treated water 50 μl.

[0181] Reagent Buffer for Extraction

[0182] 1M Tris HCl pH 9.0

[0183] 1% SDS

[0184] (β-mercaptoethanol 120 μl was added to 6 ml of buffer before use)

Example 2 Purification of Poly(A)+RNA and Synthesis of cDNA

[0185] Poly(A)+RNA was purified from total RNA 100 μg obtained inexample 1 by applying with Dynabeads Oligo (dT) 25 (DYNAL Inc.). Thispoly(A)+RNA was treated with reverse transcription reaction by M-MLVreverse transcriptase (TOYOBO Co. Ltd.) at 37° C. for 1 hour using thefollowing hybrid primer to obtain cDNA.

[0186] Hybrid primer (dT¹⁷ adapter primer):5′-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3′

Example 3 RT-PCR and Confirmation of Base Sequence

[0187] PCR was conducted with the primer specific to hybrid primer andthe 5′ primer of FRE1 using cDNA obtained in example 2 as a template.

[0188] Reaction product of PCR was electrophoresed with 0.8% agarosegel, and the obtained band was cloned to pT7Blue(R) vector (TakaraCorp.). Colony was shake cultured in LB medium for overnight, extractedthe plasmid by alkaline-SDS method, and the base sequence of 7 clones,to which the insertion was confirmed by restriction enzyme treatment,was determined by using Bca BEST DNA polymerase (“BiotechnologyExperiments Illustrated, Fundamentals of gene analysis”) (Shujun-Sha).Primer specific to hybrid primer: 5′-GACTCGAGTCGACATCG-3′ 5′ primer ofFRE1: 5′-ACACTTATTAGCACTTCATGTATT-3′

[0189] Reaction condition for PCR:

[0190] (1) 95° C. for 5 minutes;

[0191] (2) 95° C. for 40 seconds;

[0192] (3) 55° C. for 30 seconds;

[0193] (4) 72° C. for 1 minute;

[0194] (5) 72° C. for 10 minutes; and

[0195] (6) 4° C.

[0196] In the above procedures, (2), (3) and (4) were repeated 40 times.

[0197] As a result, in the transgenic tobacco transformed with FRE1,poly(A) was attached at the position as shown in FIG. 3, in thetranscribed mRNA from FRE1 gene.

[0198] Attached points of poly(A) were not uniform, and several lengthof mRNA was observed. A sequence, which might be recognized as poly(A)signal at the upstream of poly(A) site, was indicated as putativepoly(A) signal.

Example 4 Production of Each Segment by PCR

[0199] Each segment was prepared by PCR as illustrated in FIG. 5. Thesuper Taq (Sawady Inc.) was used as Taq polymerase.

[0200] Composition of PCR reaction is as follows.

[0201] PCR reaction solution in the first step: 10 × buffer 10 μl  2 mMdNTP mixture 10 μl 20 μM primer (−3)  5 μl 20 μM primer (−4)  5 μl

[0202] distilled water to total volume 99.5 μl

[0203] PCR reaction solution in the second step: PCR reaction solutionin the first step   1 μl 10 × buffer   10 μl  2 mM dNTP mixture   10 μl20 μM primer (−2)   5 μl 20 μM primer (−5)   5 μl distilled water tototal volume 99.5 μl

[0204] PCR reaction solution in the third step:

[0205] PCR reaction solution in the second step 1 μl PCR reactionsolution in the second step   1 μl 10 × buffer   10 μl  2 mM dNTPmixture   10 μl 20 μM primer (−1)   5 μl 20 μM primer (−6)   5 μldistilled water to total volume 99.5 μl

[0206] Reaction conditions for PCR:

[0207] (1) 95° C. for 5 minutes;

[0208] (2) add Taq 0.5 μl

[0209] (3) 95° C. for 40 seconds;

[0210] (4) 45° C. for 1 minute;

[0211] (5) 72° C. for 1 minute;

[0212] (6) 94° C. for 40 seconds;

[0213] (7) 60° C. for 30 seconds;

[0214] (8) 72° C. for 1 minute;

[0215] (9) 72° C. for 10 minutes;

[0216] (10) 4° C.

[0217] The above procedures of (3), (4) and (5) were repeated 5 times,and the procedures of (6), (7) and (8) ) were repeated 20 times,respectively.

Example 5 Cloning and Confirmation of Base Sequence

[0218] After electrophoresis of PCR reaction solution in the third stepin example 4 with 0.8% agarose gel, a band, which had expected length(417-436 bp), was cleaved and purified, then was cloned into the plasmidpT7Blue (R) vector (Takara Inc.). The base sequence of the thus obtainedclone was confirmed and the exact base sequence was selected usingSHIMADZU luminescent DNA sequencer DSQ-1000L.

[0219] After obtaining segment of each exact sequence, full length ofrefre1 was prepared as shown in FIG. 8 by applying with restrictionenzyme sites. A direction of insertion of the segment B and E wasessential for preparing the full length. As for the other segments, thesequence containing exact base sequence was used without relation to thedirection of insertion.

[0220] Full length of base sequence of the synthesized refre1 is shownin sequence listing SEQ ID NO: 1 and FIG. 9.

Example 6 Introduction of refre1 into Tobacco

[0221] A gene refre1 synthesized in example 5 was introduced intotobacco (Nicotiana tabacum L. var. SRI). As a result of transformation,68 individual plants resistant to kanamycin were generated. GenomicSouthern hybridization was performed in order to confirm introduction ofrefre1, an objective gene, in the generated plant and copying numberthereof As a result, existence of one to several copies of refre1 genewas confirmed.

[0222] A method from gene introduce into plant cells to generation ofplant was performed according to description in “Laboratory Manual forFunctional Analysis of Plant Genes” (Maruzen).

[0223] (1) Preparation of Binary Vector pRF1 for Transformation

[0224] XbaI and Sad fragments of refre1, which were cloned in pT7Blue(R) vector, were exchanged with ORF of β-glucronidase of binary vectorpBI121 to prepare a binary vector pRF1. A structure of the binary vectorpRF1 is shown in FIG. 11.

[0225] (2) Transfer of Binary Vector pRF1 into Agrobacterium

[0226]Agrobacterium tumefaciens C58 was shake cultured at 26° C. for 2nights in LB liquid medium 1 ml containing suitable antibiotic, and E.coli having pRF1 and E. coli having helper plasmid pRK2013 were shakecultured at 37° C. for one night in LB liquid medium 1 ml containingsuitable antibiotic. Each 100 μl was mixed on the LB plate withoutcontaining antibiotics. After the mixture was cultured at 26° C. for 2nights, plate surface was scraped out the plate using platinum loop andincubated to form single colony on the selection plate [LB platecontaining 100 μg/μl rifampicin (Rf) and 25 μg/μl kanamycin (Km)] (at26° C. for 2 nights).

[0227] The thus obtained single colony was shake cultured in LB (Km andRf) liquid medium 4 ml at 26° C. for 2 nights, and the plasmid wasextracted by alkaline-SDS method, then existence of pRF1 was confirmedby observing cleavage patterns by restriction enzymes.

[0228] (3) Infection of Agrobacterium to Tobacco and Regeneration ofPlant

[0229] Two or three young leaves of tobacco (Nicotiana tabacum L. var.SRI), size about 8 cm, were cut, put them into the petri dish filledwith sterilized water (hypochlorous acid 10% and Tween 20, 0.1%), andsterilized with stirring for 15 minutes. The leaves were rinsed withsterilized water for 3 times, and were cut in 8 mm square using scalpel.Cultured liquid of Agrobacterium 3 ml having binary vector pRF1 culturedat 26° C. for 2 nights was added to fragments of leaves in the petridish.

[0230] After one minute, the liquid was immediately removed off by usingPasture pipette and the residual liquid was removed off using autoclavedsterilized filter paper. The leaves were put on the MS medium (II)hereinbelow and cultured at 25° C. for 3 days under lighting condition.Thereafter fragments of leaves were transferred to MS medium (III) andcultured for 1 week, then transferred to the MS medium (IV) andsubcultured in every 2 weeks. When calli were induced and shoots wereformed, the shoots were cut using scalpel and transferred to the MSmedium (V). The shoots with roots were inoculated to vermiculite, andraised with supplying hyponex (Hyponex Japan Co., Ltd) to obtain theregenerated plant.

[0231] The compositions of MS medium for tobacco used in the experimentshereinbefore are as follows.

[0232] Major elements (g/l) NH₄NO₃ 1.65 KNO₃ 1.9 CaCl₂.2H₂O 0.44MgSO₄.7H₂O 0.44 KH₂PO₄ 0.17

[0233] Minor elements (mg/l) H₃BO₄ 6.2 MnSO₄.4H₂O 22.3 ZnSO₄.7H₂O 8.6 KI0.83 Na₂MoO₄.2H₂O 0.25 CuSO₄.5H₂O 0.025 CoCl₂.6H₂O 0.025 Fe(III)Na-EDTA0.042 mg/l myo-inositol 100 mg/l thiamine 5 mg/l sucrose 30 g/lgeranylated g 2 g/l

[0234] MS medium (I) was prepared by the composition hereinbefore. Theother MS media were prepared by adding the following phytohormone and/orantibiotics to the MS medium (1). Phytohormone benzyladenine (BA)  1.0mg/l naphthaleneacetic acid (NAA)  0.1 mg/l Antibiotics kanamycin  100mg/l claforan  200 mg/l

[0235] MS medium (I) MS medium (I)+BA+NAA

[0236] MS medium (III) MS medium (I)+BA+NAA+claforan

[0237] MS medium (V) MS medium (I)+BA+NAA+claforan+kanamycin

[0238] MS medium (V) MS medium (I)+kanamycin

Example 7 Southern Analysis

[0239] (1) Extraction of Genomic DNA from Tobacco

[0240] Extraction of genomic DNA from tobacco was performed according tothe method described in “Plant Cell Engineering Series: Protocol for PCRExperiments in Plants” (Shujun-Sha).

[0241] Leaves 0.1-0.2 g were put in the mortar, and liquid nitrogen wasadded thereto, then leaves were completely mashed. The crushed leaveswere put into the Eppendorf tube, and 2% CTAB solution 300 μl was addedand mixed, then treated at 65° C. for 30 minutes. Equal amount ofchloroform and isoamyl alcohol (24:1) was added and mixed for 5 minutes.

[0242] The mixture was centrifuged at 12000 rpm for 15 minutes, and theupper layer was transferred to the new tube, then chloroform-isoamylalcohol extraction was repeated once again, and the upper layer wastransferred to the new tube. 1-1.5 volume of 1% CTAB solution was added,mixed, allowed standing at room temperature for 1 hour, and centrifugedat 8000 rpm for 10 minutes. The upper layer was discarded and 1M CsCl400 μl was added to the residue, and heated at 65° C. until completedissolving the precipitate. 100% ethanol 800 μl was added thereto,mixed, allowed to standing at −20° C. for 20 minutes, then centrifugedat 12000 rpm for 5 minutes. The upper layer was discarded, and theresidue was washed with 70% ethanol, dried in vacuum and dissolved in TEbuffer 30 μl.

[0243] Reagents 2% CTAB Solution Tris-HCl (pH 8.0)  100 mM EDTA (pH 8.0)  20 mM NaCl  1.4 M

[0244] CTAB (cetyltrimethylammonium bromide) 2%

[0245] 1% CTAB solution Tris-HCl (pH 8.0) 50 mM EDTA (pH 8.0) 20 mM CTAB1%

[0246] (2) Cleavage of Genomic DNA by Restriction Enzyme andElectrophoresis

[0247] Restriction enzyme treatments were performed by digestion usingEcoRI and HindIII, by which sequence from pCaMV35S to tNOS was cleaved,and by digestion using only HindIII, by which sequence of upstream ofpCaMV35S was cleaved.

[0248] Genomic DNA 10 μg with the reaction volume 100 μl was treated byrestriction enzyme for overnight, precipitated by adding ethanol anddissolved the precipitate in TE buffer 20 μl. To the solution was addedthe loading buffer 2 μl, and the solution was electrophoresed with 0.8%agarose gel at 60V for 5 hours. After completion of electrophoresis, gelwas stained with ethidium bromide and photographed on the UVtransluminater with the scale.

[0249] (3) Blotting and Hybridization

[0250] Gel after photographing was washed with distilled water, and wasshaken in 0.2 N HCl for 10 minutes. A method of blotting was performedaccording to the description in “Cloning and Sequencing”(Noson-Bunka-Sha). The gel was transferred to nylon membrane (NewHybond-N+Amersham) with 0.4 N NaOH, and the membrane washed with 2×SSPEfor 5 minutes, and dried at room temperature for 3 hours. A method ofhybridization was referred with “Biotechnology Experiments Illustrated,Fundamentals of Gene Analysis” (Shujun-Sha) The membrane was treated forprehybridization with prehybridization buffer 30 ml, which waspreviously warmed at 65° C., for 1 hour at 65° C., and the hybridizationbuffer was exchanged (25 ml). Probe was added and hybridization wasperformed at 65° C. for 12 hours. The membrane was washed with washingsolution, which was previously warmed at 65° C., twice at 65° C. for 10minutes, and was washed once with high stringent washing solution at 65°C. for 10 minutes. The membrane was wrapped with Saran wrap, exposed onimaging plate for 24 hours, and result was confirmed by image analyzer(Fuji Photo Film Co. Ltd.).

[0251] Reagents

[0252] 20×SSPE NaCl   3 M NaH₂PO₄ 0.2 M EDTA   1 mM

[0253] 1M Church phosphate buffer

[0254] NaHPO₄ 0.5 mol was added to distilled water about 800 ml,adjusted pH to 7.2 by H₃PO₄, then filled up to 1 liter by addingdistilled water, and autoclaved.

[0255] Hybridization Buffer Church phosphate buffer 0.5 M EDTA   1 mMSDS (v/v) 7%

[0256] Denatured salmon sperm (1 mg/ml) {fraction (1/100)} vol. wasadded before use.

[0257] Washing Solution Church phosphate buffer 40 mM SDS (v/v)  1%

[0258] High Stringent Washing Solution

[0259] 0.2×SSPE SDS (v/v) 0.1%

[0260] (4) Preparation of Probe

[0261] Probe was prepared by random primer DNA labeling kit ver. 2.0(Takara Corp.) using full length refre1 as a template (proviso that[α-³²P]-dATP was used), and non-reacted [α-³²P]-dATP was removed usingProbe Quant TM G-50 Micro Columns (Pharmacia, Biotech Inc.).

[0262] Result is shown in FIG. 14. The left side in FIG. 14 showsdigestion using restriction enzymes EcoRI and HindIII, and the rightside in FIG. 14 shows digestion using only HindIII. In FIG. 14, W.T.means wild type.

Example 8 Northern Analysis

[0263] (1) Extraction of Total RNA

[0264] Total RNA was extracted from leaves of the transgenic tobacco, towhich refre1 gene was introduced, and leaves of the wilt type tobaccoaccording to the same method as described in example 1.

[0265] (2) Electrophoresis of RNA

[0266] Electrophoresis vessel, gel receiver, comb and Erlenmeyer flaskwere treated previously with abSolve (RNase inhibitor, Du Pont Inc.).20×MOPS 10 ml, agarose 2.4 g and sterilized distilled water 100 ml werepoured into Erlenmeyer flask, and agarose was dissolved using microwaveoven. Formaldehyde 10 ml was added to the gel which was cooled to about50° C., and sterilized distilled water was added up to 200 ml, which wasused when gelification occurred. 1×MOPS about 800 ml was added in theelectrophoresis vessel, and added ethidium bromide 10 mg/ml thereto foruse as electrophoresis buffer. RNA sample buffer 16 μl was added to thetotal RNA 10 μg, filled up to 20 μl with sterilized distilled water, andthe mixture was warmed at 65° C. for 10 minutes, then allowed tostanding for 5 minutes on ice, and was electrophoresed. Electrophoresiscondition was that after electrophoresis was performed at 60 V for 1hour, further electrophoresis was performed at 120 V for 2 hours.

[0267] Reagents:

[0268] 20×MOPS MOPS  0.4 M NaOAc  0.1 M EDTA 0.02 M

[0269] RNA sample buffer Formaldehyde 1.6 ml Formamide 5.0 ml 20 × MOPS0.5 ml glycerol pigment solution 1.6 ml Total 8.7 ml

[0270] Glycerol pigment solution glycerol   5 ml bromophenol blue   1 mgxylenecyanol   1 mg 0.5 M EDTA (pH 8.0) 0.02 ml

[0271] (3) Blotting and Hybridization

[0272] After electrophoresis, gel was set on UV illuminator andphotographed with the scale. A method of blotting was followed accordingto the description in “Cloning and Sequencing” (Noson-Bunka-Sha).Namely, RNA was transferred from gel to nylon membrane (New Hybond-N,Amersham Inc.) with 20×SSPE. After 12 hours, the membrane was washedwith 2×SSPE for 5 minutes, and dried at room temperature for 3 hours,then RNA was fixed on the membrane by irradiating with UV for 5 minutes.

[0273] A method of hybridization was performed as same as the case ofSouthern analysis.

[0274] Result is shown in FIG. 15. In FIG. 15, W.T. indicates wild type.No band was detected in the lane of wild type (W.T.). In No. 1 and No. 2lanes, major band was detected at the size of 2.5 kb, and several bandswere detected in the lower position thereof.

Example 9 Confirmation of Ferric-ehelate Reductase

[0275] The transgenic tobacco, to which refre1 gene was introduced, andwild type tobacco were transplanted in vermiculite and raised withsupplying hyponex. Ferric-chelate reductase activity was confirmed byusing plants, about 5 cm-10 cm.

[0276] For confirmation of ferric-chelate reductase activity, redcoloring generated by formation of complex with bathophenanthrolinedisulfonic acid (BPDS), which was strong chelating agent for Fe(II), andFe(II) was applied. Agarose was added to assay buffer up to 0.4%,dissolved by using microwave oven, and cooled. 500 μM Fe(III)-EDTA,{fraction (1/100)} vol., and 500 μM BPDS, {fraction (1/100)} vol., wereadded to the slightly cooled gel, and stirred to put in the vessel, thenwaited for solidification. After removed off vermiculite from thetransformant and wild type tobacco, roots were laid on the gel, andshielded from light and allowed to standing at 27° C. for 24 hours.

[0277] The similar experiment was performed using second generation ofthe transgenic plant, which was germinated from seeds of the regeneratedplant. Reaction time in this experiment was set for 1 hour.

[0278] Assay Buffer CaSO₄ 0.2 mM MES buffer pH 5.5 5.0 mM

[0279] Photographs showing confirmation of ferric-chelate reductaseactivity are shown in FIG. 16 and FIG. 17. Photograph showingconfirmation of ferric-chelate reductase activity of the secondgeneration plant is shown in FIG. 18. Reduction of Fe(III) was confirmedas a result of coloring of the transformant in the rhizosphere.

1 38 1 2092 DNA Artificial Sequence Description of Artificial SequenceSynthetic saccharomyces cerevisiae 1 gaattctcta gactccacc atg gtt agaacc aga gtc ctt ttc tgc ctc ttc 52 Met Val Arg Thr Arg Val Leu Phe CysLeu Phe 1 5 10 atc tct ttc ttc gct aca gtc caa tcg agc gct aca ctc atctcc act 100 Ile Ser Phe Phe Ala Thr Val Gln Ser Ser Ala Thr Leu Ile SerThr 15 20 25 tca tgc att tct cag gct gca ctg tac cag ttc gga tgc tca agcaag 148 Ser Cys Ile Ser Gln Ala Ala Leu Tyr Gln Phe Gly Cys Ser Ser Lys30 35 40 tca aag tct tgc tac tgc aag aac atc aat tgg ctc gga agc gtc act196 Ser Lys Ser Cys Tyr Cys Lys Asn Ile Asn Trp Leu Gly Ser Val Thr 4550 55 gca tgc gct tat gag aac tcc aaa tct aac aag act ctg gac tcc gct244 Ala Cys Ala Tyr Glu Asn Ser Lys Ser Asn Lys Thr Leu Asp Ser Ala 6065 70 75 ttg atg aaa ctt gcc agc caa tgc tca agt atc aag gtt tac aca ctg292 Leu Met Lys Leu Ala Ser Gln Cys Ser Ser Ile Lys Val Tyr Thr Leu 8085 90 gag gac atg aag aac atc tac ctt aat gca agt aac tac ctt cgc gct340 Glu Asp Met Lys Asn Ile Tyr Leu Asn Ala Ser Asn Tyr Leu Arg Ala 95100 105 cct gag aaa tcc gat aag aag aca gtt gtt tca caa ccg ttg atg gca388 Pro Glu Lys Ser Asp Lys Lys Thr Val Val Ser Gln Pro Leu Met Ala 110115 120 aat gag acg gcc tat cac tac tac tat gag gaa aac tat ggg atc cac436 Asn Glu Thr Ala Tyr His Tyr Tyr Tyr Glu Glu Asn Tyr Gly Ile His 125130 135 ttg aat ttg atg cga tct caa tgg tgc gca tgg ggc ctc gtc ttc ttc484 Leu Asn Leu Met Arg Ser Gln Trp Cys Ala Trp Gly Leu Val Phe Phe 140145 150 155 tgg gtc gca gtc ctt acc gcc gca act atc ttg aac att ctc aaacgc 532 Trp Val Ala Val Leu Thr Ala Ala Thr Ile Leu Asn Ile Leu Lys Arg160 165 170 gta ttc ggc aag aac att atg gca aat tct gtt aag aag tct cttatc 580 Val Phe Gly Lys Asn Ile Met Ala Asn Ser Val Lys Lys Ser Leu Ile175 180 185 tac cca agc gtt tac aaa gac tac aac gag aga act ttc tat ctttgg 628 Tyr Pro Ser Val Tyr Lys Asp Tyr Asn Glu Arg Thr Phe Tyr Leu Trp190 195 200 aaa cgt ttg cca ttc aac ttt aca act cga ggc aaa gga ctc gtagtt 676 Lys Arg Leu Pro Phe Asn Phe Thr Thr Arg Gly Lys Gly Leu Val Val205 210 215 ctt atc ttt gtc att ctg act att ctc tca ctc tct ttc gga cataac 724 Leu Ile Phe Val Ile Leu Thr Ile Leu Ser Leu Ser Phe Gly His Asn220 225 230 235 atc aag ttg cca cat cct tac gat aga cct aga tgg aga agatca atg 772 Ile Lys Leu Pro His Pro Tyr Asp Arg Pro Arg Trp Arg Arg SerMet 240 245 250 gca ttc gtc tca cgc cgt gct gac ttg atg gca atc gct cttttc ccc 820 Ala Phe Val Ser Arg Arg Ala Asp Leu Met Ala Ile Ala Leu PhePro 255 260 265 gtg gtg tac ctt ttc ggt atc cgg aac aac ccc ttc atc ccaatc acc 868 Val Val Tyr Leu Phe Gly Ile Arg Asn Asn Pro Phe Ile Pro IleThr 270 275 280 gga ttg agc ttt agt act ttc aac ttt tac cac aaa tgg tcagca tac 916 Gly Leu Ser Phe Ser Thr Phe Asn Phe Tyr His Lys Trp Ser AlaTyr 285 290 295 gtc tgc ttc atg tta gcc gtc gtc cat tca atc gtt atg accgct tca 964 Val Cys Phe Met Leu Ala Val Val His Ser Ile Val Met Thr AlaSer 300 305 310 315 gga gtt aaa cga gga gta ttc cag tct ctt gta agg aaattc tac ttc 1012 Gly Val Lys Arg Gly Val Phe Gln Ser Leu Val Arg Lys PheTyr Phe 320 325 330 aga tgg gga ata gta gcc aca att ctt atg tcc atc atcatt ttc cag 1060 Arg Trp Gly Ile Val Ala Thr Ile Leu Met Ser Ile Ile IlePhe Gln 335 340 345 tcc gag aag gtc ttc agg aac cga ggt tat gaa atc ttctta ctt att 1108 Ser Glu Lys Val Phe Arg Asn Arg Gly Tyr Glu Ile Phe LeuLeu Ile 350 355 360 cac aaa gcc atg aac atc atg ttt atc ata gct atg tattac cat tgc 1156 His Lys Ala Met Asn Ile Met Phe Ile Ile Ala Met Tyr TyrHis Cys 365 370 375 cac aca cta gga tgg atg ggc tgg atc tgg tcc atg gctggc atc ctc 1204 His Thr Leu Gly Trp Met Gly Trp Ile Trp Ser Met Ala GlyIle Leu 380 385 390 395 tgc ttc gac agg ttc tgc cga att gta cgt atc atcatg aac gga ggt 1252 Cys Phe Asp Arg Phe Cys Arg Ile Val Arg Ile Ile MetAsn Gly Gly 400 405 410 ctt aag acc gcc act ttg tcg acc aca gat gat tctaac gtt atc aag 1300 Leu Lys Thr Ala Thr Leu Ser Thr Thr Asp Asp Ser AsnVal Ile Lys 415 420 425 atc tct gtc aag aag cct aag ttc ttc aag tat caagtg gga gca ttt 1348 Ile Ser Val Lys Lys Pro Lys Phe Phe Lys Tyr Gln ValGly Ala Phe 430 435 440 gcc tat atg tac ttt ctt tca cca aaa tca gcc tggttc tac agt ttt 1396 Ala Tyr Met Tyr Phe Leu Ser Pro Lys Ser Ala Trp PheTyr Ser Phe 445 450 455 caa tct cat ccc ttc aca gtc cta tca gaa agg cacaga gat cct aac 1444 Gln Ser His Pro Phe Thr Val Leu Ser Glu Arg His ArgAsp Pro Asn 460 465 470 475 aac cca gat caa cta act atg tac gtc aaa gctaac aag ggc att acg 1492 Asn Pro Asp Gln Leu Thr Met Tyr Val Lys Ala AsnLys Gly Ile Thr 480 485 490 aga gta ctt ctt agc aaa gtt cta agc gct ccaaac cat acc gtt gat 1540 Arg Val Leu Leu Ser Lys Val Leu Ser Ala Pro AsnHis Thr Val Asp 495 500 505 tgc aag att ttc tta gag gga cca tat ggc gtaact gtc cct cac att 1588 Cys Lys Ile Phe Leu Glu Gly Pro Tyr Gly Val ThrVal Pro His Ile 510 515 520 gcc aaa ctt aag aga aat cta gta gga gta gctgcg ggc ctc ggc gtg 1636 Ala Lys Leu Lys Arg Asn Leu Val Gly Val Ala AlaGly Leu Gly Val 525 530 535 gca gcc atc tac ccc cat ttc gta gaa tgc cttaga ttg cct agc act 1684 Ala Ala Ile Tyr Pro His Phe Val Glu Cys Leu ArgLeu Pro Ser Thr 540 545 550 555 gat caa ctg cag cac aag ttc tac tgg atcgtc aac gac ctt agt cac 1732 Asp Gln Leu Gln His Lys Phe Tyr Trp Ile ValAsn Asp Leu Ser His 560 565 570 ctt aag tgg ttc gaa aac gag cta caa tggctt aag gag aaa tct tgt 1780 Leu Lys Trp Phe Glu Asn Glu Leu Gln Trp LeuLys Glu Lys Ser Cys 575 580 585 gaa gtc tct gtc atc tac act ggg tca tcagtg gag gat aca aac tca 1828 Glu Val Ser Val Ile Tyr Thr Gly Ser Ser ValGlu Asp Thr Asn Ser 590 595 600 gat gag tcc act aag ggt ttc gat gac aaggaa gaa tct gaa atc acc 1876 Asp Glu Ser Thr Lys Gly Phe Asp Asp Lys GluGlu Ser Glu Ile Thr 605 610 615 gta gaa tgc ctt aac aag agg cca gac ctcaaa gag cta gtg aga tca 1924 Val Glu Cys Leu Asn Lys Arg Pro Asp Leu LysGlu Leu Val Arg Ser 620 625 630 635 gag atc aaa ttg tca gaa ctc gag aacaac aac atc act ttc tac tca 1972 Glu Ile Lys Leu Ser Glu Leu Glu Asn AsnAsn Ile Thr Phe Tyr Ser 640 645 650 tgc gga cca gcg act ttc aat gac gacttt agg aat gca gtt gta caa 2020 Cys Gly Pro Ala Thr Phe Asn Asp Asp PheArg Asn Ala Val Val Gln 655 660 665 ggt atc gat tct agt ctg aag ata gatgtc gaa cta gag gag gag agt 2068 Gly Ile Asp Ser Ser Leu Lys Ile Asp ValGlu Leu Glu Glu Glu Ser 670 675 680 ttt act tgg taagagctca agctt 2092Phe Thr Trp 685 2 686 PRT Artificial Sequence Description of ArtificialSequence Synthetic saccharomyces cerevisiae 2 Met Val Arg Thr Arg ValLeu Phe Cys Leu Phe Ile Ser Phe Phe Ala 1 5 10 15 Thr Val Gln Ser SerAla Thr Leu Ile Ser Thr Ser Cys Ile Ser Gln 20 25 30 Ala Ala Leu Tyr GlnPhe Gly Cys Ser Ser Lys Ser Lys Ser Cys Tyr 35 40 45 Cys Lys Asn Ile AsnTrp Leu Gly Ser Val Thr Ala Cys Ala Tyr Glu 50 55 60 Asn Ser Lys Ser AsnLys Thr Leu Asp Ser Ala Leu Met Lys Leu Ala 65 70 75 80 Ser Gln Cys SerSer Ile Lys Val Tyr Thr Leu Glu Asp Met Lys Asn 85 90 95 Ile Tyr Leu AsnAla Ser Asn Tyr Leu Arg Ala Pro Glu Lys Ser Asp 100 105 110 Lys Lys ThrVal Val Ser Gln Pro Leu Met Ala Asn Glu Thr Ala Tyr 115 120 125 His TyrTyr Tyr Glu Glu Asn Tyr Gly Ile His Leu Asn Leu Met Arg 130 135 140 SerGln Trp Cys Ala Trp Gly Leu Val Phe Phe Trp Val Ala Val Leu 145 150 155160 Thr Ala Ala Thr Ile Leu Asn Ile Leu Lys Arg Val Phe Gly Lys Asn 165170 175 Ile Met Ala Asn Ser Val Lys Lys Ser Leu Ile Tyr Pro Ser Val Tyr180 185 190 Lys Asp Tyr Asn Glu Arg Thr Phe Tyr Leu Trp Lys Arg Leu ProPhe 195 200 205 Asn Phe Thr Thr Arg Gly Lys Gly Leu Val Val Leu Ile PheVal Ile 210 215 220 Leu Thr Ile Leu Ser Leu Ser Phe Gly His Asn Ile LysLeu Pro His 225 230 235 240 Pro Tyr Asp Arg Pro Arg Trp Arg Arg Ser MetAla Phe Val Ser Arg 245 250 255 Arg Ala Asp Leu Met Ala Ile Ala Leu PhePro Val Val Tyr Leu Phe 260 265 270 Gly Ile Arg Asn Asn Pro Phe Ile ProIle Thr Gly Leu Ser Phe Ser 275 280 285 Thr Phe Asn Phe Tyr His Lys TrpSer Ala Tyr Val Cys Phe Met Leu 290 295 300 Ala Val Val His Ser Ile ValMet Thr Ala Ser Gly Val Lys Arg Gly 305 310 315 320 Val Phe Gln Ser LeuVal Arg Lys Phe Tyr Phe Arg Trp Gly Ile Val 325 330 335 Ala Thr Ile LeuMet Ser Ile Ile Ile Phe Gln Ser Glu Lys Val Phe 340 345 350 Arg Asn ArgGly Tyr Glu Ile Phe Leu Leu Ile His Lys Ala Met Asn 355 360 365 Ile MetPhe Ile Ile Ala Met Tyr Tyr His Cys His Thr Leu Gly Trp 370 375 380 MetGly Trp Ile Trp Ser Met Ala Gly Ile Leu Cys Phe Asp Arg Phe 385 390 395400 Cys Arg Ile Val Arg Ile Ile Met Asn Gly Gly Leu Lys Thr Ala Thr 405410 415 Leu Ser Thr Thr Asp Asp Ser Asn Val Ile Lys Ile Ser Val Lys Lys420 425 430 Pro Lys Phe Phe Lys Tyr Gln Val Gly Ala Phe Ala Tyr Met TyrPhe 435 440 445 Leu Ser Pro Lys Ser Ala Trp Phe Tyr Ser Phe Gln Ser HisPro Phe 450 455 460 Thr Val Leu Ser Glu Arg His Arg Asp Pro Asn Asn ProAsp Gln Leu 465 470 475 480 Thr Met Tyr Val Lys Ala Asn Lys Gly Ile ThrArg Val Leu Leu Ser 485 490 495 Lys Val Leu Ser Ala Pro Asn His Thr ValAsp Cys Lys Ile Phe Leu 500 505 510 Glu Gly Pro Tyr Gly Val Thr Val ProHis Ile Ala Lys Leu Lys Arg 515 520 525 Asn Leu Val Gly Val Ala Ala GlyLeu Gly Val Ala Ala Ile Tyr Pro 530 535 540 His Phe Val Glu Cys Leu ArgLeu Pro Ser Thr Asp Gln Leu Gln His 545 550 555 560 Lys Phe Tyr Trp IleVal Asn Asp Leu Ser His Leu Lys Trp Phe Glu 565 570 575 Asn Glu Leu GlnTrp Leu Lys Glu Lys Ser Cys Glu Val Ser Val Ile 580 585 590 Tyr Thr GlySer Ser Val Glu Asp Thr Asn Ser Asp Glu Ser Thr Lys 595 600 605 Gly PheAsp Asp Lys Glu Glu Ser Glu Ile Thr Val Glu Cys Leu Asn 610 615 620 LysArg Pro Asp Leu Lys Glu Leu Val Arg Ser Glu Ile Lys Leu Ser 625 630 635640 Glu Leu Glu Asn Asn Asn Ile Thr Phe Tyr Ser Cys Gly Pro Ala Thr 645650 655 Phe Asn Asp Asp Phe Arg Asn Ala Val Val Gln Gly Ile Asp Ser Ser660 665 670 Leu Lys Ile Asp Val Glu Leu Glu Glu Glu Ser Phe Thr Trp 675680 685 3 17 DNA Artificial Sequence Description of Artificial SequencePrimer 3 gactcgagtc gacatcg 17 4 24 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 4 acacttatta gcacttcatg tatt 24 5 83 DNAArtificial Sequence Description of Artificial Sequence Primer 5gaattctcta gactccacca tggttagaac cagagtcctt ttctgcctct tcatctcttt 60cttcgctaca gtccaatcga gcg 83 6 83 DNA Artificial Sequence Description ofArtificial Sequence Primer 6 gtccaatcga gcgctacact catctccact tcatgcatttctcaggctgc actgtaccag 60 ttcggatgct caagcaagtc aaa 83 7 83 DNAArtificial Sequence Description of Artificial Sequence Primer 7caagcaagtc aaagtcttgc tactgcaaga acatcaattg gctcggaagc gtcactgcat 60gcgcttatga gaactccaaa tct 83 8 83 DNA Artificial Sequence Description ofArtificial Sequence Primer 8 tccagtgtgt aaaccttgat acttgagcat tggctggcaagtttcatcaa agcggagtcc 60 agagtcttgt tagatttgga gtt 83 9 83 DNAArtificial Sequence Description of Artificial Sequence Primer 9tgtcttctta tcggatttct caggagcgcg aaggtagtta cttgcattaa ggtagatgtt 60cttcatgtcc tccagtgtgt aaa 83 10 83 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 10 ggatcccata gttttcctca tagtagtagtgataggccgt ctcatttgcc atcaacggtt 60 gtgaaacaac tgtcttctta tcg 83 11 80DNA Artificial Sequence Description of Artificial Sequence Primer 11ggatccactt gaatttgatg cgatctcaat ggtgcgcatg gggcctcgtc ttcttctggg 60tcgcagtcct taccgccgca 80 12 80 DNA Artificial Sequence Description ofArtificial Sequence Primer 12 ccttaccgcc gcaactatct tgaacattctcaaacgcgta ttcggcaaga acattatggc 60 aaattctgtt aagaagtctc 80 13 80 DNAArtificial Sequence Description of Artificial Sequence Primer 13gttaagaagt ctcttatcta cccaagcgtt tacaaagact acaacgagag aactttctat 60ctttggaaac gtttgccatt 80 14 80 DNA Artificial Sequence Description ofArtificial Sequence Primer 14 agagtgagag aatagtcaga atgacaaagataagaactac gagtcctttg cctcgagttg 60 taaagttgaa tggcaaacgt 80 15 80 DNAArtificial Sequence Description of Artificial Sequence Primer 15aatgccattg atcttctcca tctaggtcta tcgtaaggat gtggcaactt gatgttatgt 60ccgaaagaga gtgagagaat 80 16 80 DNA Artificial Sequence Description ofArtificial Sequence Primer 16 tccggatacc gaaaaggtac accacggggaaaagagcgat tgccatcaag tcagcacggc 60 gtgagacgaa tgccattgat 80 17 83 DNAArtificial Sequence Description of Artificial Sequence Primer 17tccggaacaa ccccttcatc ccaatcaccg gattgagctt tagtactttc aacttttacc 60acaaatggtc agcatacgtc tgc 83 18 83 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 18 gcatacgtct gcttcatgtt agccgtcgtccattcaatcg ttatgaccgc ttcaggagtt 60 aaacgaggag tattccagtc tct 83 19 83DNA Artificial Sequence Description of Artificial Sequence Primer 19tattccagtc tcttgtaagg aaattctact tcagatgggg aatagtagcc acaattctta 60tgtccatcat cattttccag tcc 83 20 83 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 20 ataaacatga tgttcatggc tttgtgaataagtaagaaga tttcataacc tcggttcctg 60 aagaccttct cggactggaa aat 83 21 83DNA Artificial Sequence Description of Artificial Sequence Primer 21gaggatgcca gccatggacc agatccagcc catccatcct agtgtgtggc aatggtaata 60catagctatg ataaacatga tgt 83 22 83 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 22 gtcgacaaag tggcggtctt aagacctccgttcatgatga tacgtacaat tcggcagaac 60 ctgtcgaagc agaggatgcc agc 83 23 82DNA Artificial Sequence Description of Artificial Sequence Primer 23gtcgaccaca gatgattcta acgttatcaa gatctctgtc aagaagccta agttcttcaa 60gtatcaagtg ggagcatttg cc 82 24 82 DNA Artificial Sequence Description ofArtificial Sequence Primer 24 ggagcatttg cctatatgta ctttctttcaccaaaatcag cctggttcta cagttttcaa 60 tctcatccct tcacagtcct at 82 25 82DNA Artificial Sequence Description of Artificial Sequence Primer 25ttcacagtcc tatcagaaag gcacagagat cctaacaacc cagatcaact aactatgtac 60gtcaaagcta acaagggcat ta 82 26 82 DNA Artificial Sequence Description ofArtificial Sequence Primer 26 cctctaagaa aatcttgcaa tcaacggtatggtttggagc gcttagaact ttgctaagaa 60 gtactctcgt aatgcccttg tt 82 27 82DNA Artificial Sequence Description of Artificial Sequence Primer 27ggcccgcagc tactcctact agatttctct taagtttggc aatgtgaggg acagttacgc 60catatggtcc ctctaagaaa at 82 28 82 DNA Artificial Sequence Description ofArtificial Sequence Primer 28 ctgcagttga tcagtgctag gcaatctaaggcattctacg aaatgggggt agatggctgc 60 cacgccgagg cccgcagcta ct 82 29 77DNA Artificial Sequence Description of Artificial Sequence Primer 29ctgcagcaca agttctactg gatcgtcaac gaccttagtc accttaagtg gttcgaaaac 60gagctacaat ggcttaa 77 30 77 DNA Artificial Sequence Description ofArtificial Sequence Primer 30 acaatggctt aaggagaaat cttgtgaagtctctgtcatc tacactgggt catcagtgga 60 ggatacaaac tcagatg 77 31 77 DNAArtificial Sequence Description of Artificial Sequence Primer 31caaactcaga tgagtccact aagggtttcg atgacaagga agaatctgaa atcaccgtag 60aatgccttaa caagagg 77 32 77 DNA Artificial Sequence Description ofArtificial Sequence Primer 32 gtgatgttgt tgttctcgag ttctgacaatttgatctctg atctcactag ctctttgagg 60 tctggcctct tgttaag 77 33 77 DNAArtificial Sequence Description of Artificial Sequence Primer 33cgataccttg tacaactgca ttcctaaagt cgtcattgaa agtcgctggt ccgcatgagt 60agaaagtgat gttgttg 77 34 77 DNA Artificial Sequence Description ofArtificial Sequence Primer 34 aagcttgagc tcttaccaag taaaactctcctcctctagt tcgacatcta tcttcagact 60 agaatcgata ccttgta 77 35 35 DNAArtificial Sequence Description of Artificial Sequence Primer 35gactcgagtc gacatcgatt tttttttttt ttttt 35 36 2059 DNA Saccharomycescerevisiae 36 atggttagaa cccgtgtatt attctgctta tttatatctt tttttgctacggttcaatcg 60 agtgctacac ttattagcac ttcatgtatt tcccaagctg cgctataccaatttggatgt 120 tctagtaaat ctaaaagttg ctactgtaaa aacatcaatt ggctgggttcagtgacagca 180 tgtgcctatg agaattccaa atctaacaaa acactagaca gcgccttaatgaagttagca 240 tcccaatgtt caagcatcaa agtttatact ttagaggaca tgaagaatatttatttaaat 300 gcgtcaaatt atttgagagc acctgagaaa agtgataaaa aaaccgtggttagtcaaccg 360 ctcatggcga acgagacagc gtatcattat tattatgagg aaaattatggtatccatctt 420 aacctaatgc gctctcaatg gtgcgcttgg ggtctcgtct tcttctgggtgggtgtgctt 480 actgcagcca ctatcttgaa cattctgaaa agggtgtttg gtaagaacatcatggcaaac 540 tccgtcaaaa aatcacttat ttatccttct gtttacaaag attataatgaacgaactttt 600 tatttatgga agcgtctacc atttaatttt acaactcgag gcaagggtctcgtcgtatta 660 atttttgtta ttttgactat attatctctc agttttggtc ataatattaaacttccacac 720 ccatatgata ggcccagatg gagaagaagt atggcctttg tgagtcgtagagcagacttg 780 atggccattg cacttttccc agtagtctat ctattcggaa taagaaataatcccttcatc 840 cctataacag ggctttcctt ttctacattt aatttctatc ataaatggtctgcctacgtt 900 tgtttcatgt tggccgttgt acactcaatt gtcatgaccg cctcgggagtgaaaagaggt 960 gtgtttcaaa gtctggttag gaaattttac tttaggtggg gtatagtggcaacgatatta 1020 atgtctatta ttattttcca aagtgaaaaa gtatttagaa atagagggtatgagatattc 1080 cttcttattc ataaagcgat gaatattatg ttcattattg ccatgtactaccattgtcac 1140 accctgggct ggatgggttg gatttggtca atggctggta ttttatgctttgatagattc 1200 tgcaggattg ttagaataat catgaatggt ggcttgaaaa ctgctactttgagtaccact 1260 gatgattcta atgttattaa aatttcagta aaaaaaccaa agtttttcaagtaccaagta 1320 ggagctttcg catacatgta tttcttatca ccaaaaagtg catggttctatagtttccaa 1380 tcacatccat ttacagtatt atcggaacga caccgtgatc caaacaatccagatcaattg 1440 acgatgtacg taaaggcaaa taaaggtatc actcgagttt tgttatcgaaagttctaagt 1500 gctccaaatc atactgttga ttgtaaaata ttccttgaag gcccatatggtgtaacggtt 1560 ccacatatcg ctaagctaaa aagaaatctg gtaggtgtag ccgctggtttgggtgttgcg 1620 gctatttatc cgcactttgt cgaatgttta cggttaccat ctactgatcaacttcagcat 1680 aaattttact ggattgttaa tgacctatcc catttgaaat ggtttgaaaatgaattgcaa 1740 tggttaaagg agaaaagttg tgaagtctca gtcatatata ctggttccagtgttgaggac 1800 acaaattcag atgagagtac aaaaggtttt gatgataaag aagaaagcgaaatcactgtt 1860 gaatgtctca ataaaagacc tgatttgaaa gaactagtgc gctcggaaataaaactctca 1920 gaactagaga ataataatat taccttttat tcctgcgggc cagcaacgtttaacgacgat 1980 tttagaaatg cagtggtcca aggtatagac tcttccttga agattgacgttgaactagaa 2040 gaagaaagtt ttacatggt 2059 37 180 DNA Saccharomycescerevisiae CDS (1)..(180) 37 tcc gtc aaa aaa tca ctt att tat cct tct gtttac aaa gat tat aat 48 Ser Val Lys Lys Ser Leu Ile Tyr Pro Ser Val TyrLys Asp Tyr Asn 1 5 10 15 gaa cga act ttt tat tta tgg aag cgt cta ccattt aat ttt aca act 96 Glu Arg Thr Phe Tyr Leu Trp Lys Arg Leu Pro PheAsn Phe Thr Thr 20 25 30 cga ggc aag ggt ctc gtc gta tta att ttt gtt attttg act ata tta 144 Arg Gly Lys Gly Leu Val Val Leu Ile Phe Val Ile LeuThr Ile Leu 35 40 45 tct ctc agt ttt ggt cat aat att aaa ctt cca cac 180Ser Leu Ser Phe Gly His Asn Ile Lys Leu Pro His 50 55 60 38 60 PRTSaccharomyces cerevisiae 38 Ser Val Lys Lys Ser Leu Ile Tyr Pro Ser ValTyr Lys Asp Tyr Asn 1 5 10 15 Glu Arg Thr Phe Tyr Leu Trp Lys Arg LeuPro Phe Asn Phe Thr Thr 20 25 30 Arg Gly Lys Gly Leu Val Val Leu Ile PheVal Ile Leu Thr Ile Leu 35 40 45 Ser Leu Ser Phe Gly His Asn Ile Lys LeuPro His 50 55 60

1. A method for transforming a useful plant by introducing a gene ofanother species into the useful plant wherein the region of a factorrelating to the poly (A) addition of the mRNA of the useful plant to betransformed contained in the base sequence of the gene of the otherspecies is modified into another base sequence not relating to the poly(A) addition of the mRNA without substantially altering the function ofthe protein encoded by the gene to be introduced.
 2. The methodaccording to claim 1, wherein the gene of another species to beintroduced is derived from yeast.
 3. The method according to claim 1 or2, wherein the region of a factor relating to the poly (A) addition ofthe mRNA is a base sequence having AATAAA like sequence.
 4. The methodaccording to claim 3, wherein the region of a factor relating to thepoly (A) addition of the mRNA is located in a downstream from theGT-rich base sequence.
 5. The method according to any one of claims 1-4,wherein the modification of base sequence in the region of a factorrelating to the poly (A) addition of the mRNA is performed based on acodon usage of the useful higher plant to be transformed.
 6. The methodaccording to any one of claims 1-5, wherein the modification of basesequence is performed so that the region rich in base G and base T isreduced.
 7. The method according to any one of claims 1-6, wherein themodification of base sequence comprises small difference between base Gand base C covering throughout the region of gene to be introduced. 8.The method according to any one of claims 1-7, wherein the modificationof base sequence is performed so as not to have ATTTA sequence.
 9. Themethod according to any one of claims 1-8, characterized by having Kozaksequence in the upstream of the initiation codon of the gene to beintroduced.
 10. The method according to any one of claims 1-9, whereinthe gene to be introduced encodes a protein involved in absorption ofnutrition.
 11. The method according to claim 10, wherein the gene to beintroduced is the gene encoding ferric-chelate reductase FRE1.
 12. Themethod according to claim 11, wherein the gene encoding ferric-chelatereductase FRE1 is derived from yeast.
 13. The method according to anyone of claims 1-12, wherein the useful plant is grass.
 14. The methodaccording to any one of claims 1-12, wherein the useful plant istobacco.
 15. A transformed useful plant which can be produced by themethod according to claims 1-14.
 16. The plant according to claim 15,wherein the plant is seed.
 17. A nucleic acid having modified basesequence which can be used by the method according to any one of claims1-14.
 18. The nucleic acid according to claim 17, wherein the nucleicacid is DNA.
 19. The DNA according to claim 18, wherein the gene to beintroduced is the DNA encoding ferric-chelate reductase FRE1.
 20. TheDNA according to claim 19, wherein the DNA has a base sequence of SEQ IDNO:1.
 21. A method for producing the nucleic acid according to any oneof claims 17-20, wherein the nucleic acid is cleaved into severalfragments and these fragments are ligated.